The art of fermentation, i.e. the transformation of organic compounds with the aid of enzymes produced by microorganisms, is well known. Microbial activity is fairly well understood in the food industry. Fermentation is used widely in the production of alcoholic beverages, dairy products and some Oriental fermented foods, especially in tropical climates.
About one hundred years ago, Metchnikoff developed a theory that the ingestion of soured milk could improve colonic microflora through the reduction of the “auto-intoxication effect” of the colon. Today, this concept has been improved, and this field is now known as probiotics and prebiotics, defined as “a live microbial food supplement, that beneficially effects the host animal by improving its intestinal microbial balance” and “non-digestible food ingredients that benefit the host by selectively stimulating the growth or activity of one or a number of bacteria in the colon”. A “symbiotic” is a combination of probiotics and prebiotics that “beneficially effects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract by selectively stimulating the growth of, and/or by activating the metabolism of, one or a number of health promoting bacteria”.
The bacterial genera most often used in the field of probiotics are lactic acid bacteria, particularly Lactobacillus sp. and Bifidobacterium sp., these bacteria being important members of the gastrointestinal microflora of man and animals. Other microorganisms used as probiotics in humans include Escherichia coli, Streptococcus sp., Enterococcus sp., Bacteroides sp., Bacillus sp., Propionibacterium sp. and various fungi.
The external and internal surfaces of a human body are covered with bacteria. These organisms are traditionally referred to as “normal (friendly) flora”, or symbionts with commensals. These friendly bacteria are involved in dynamic bio-film communications on the skin, mouth, naso-pharyngeal, intestinal and urogenital tracts, where the appropriate microflora exists. The human body depends on this friendly microflora: it helps in food digesting, produces vital vitamins and protects against various pathogens. The mechanisms by which probiotic microorganisms provide benefits for the internal and external surfaces of the host are numerous: competing with pathogens for food, preventing the adhesion of pathogens, antimicrobial activity, colonization resistance, various immune effects, adjuvant effect, antimutagenic effects, antigenotoxic effects, influence on enzyme activity, enzyme delivery and many others.
It is accepted that probiotics are usually targeted for use in intestinal disorders. The effectiveness of probiotics has been demonstrated in the prevention and treatment of a diverse spectrum of gastrointestinal disorders, such as antibiotic-associated diarrhea, infectious bacterial and viral diarrhea, etc. Some evidence suggests a role for probiotics in reducing the risk of colon cancer and the regression of tumors. For example, U.S. Pat. No. 5,308,615 by DeLoach and U.S. Pat. No. 5,478,557 by Nisbet describe a probiotic used for control of salmonella. Also, probiotics have been used therapeutically to lower cholesterol, to reduce blood pressure, treat rheumatoid arthritis, prevent cancer, and prevent or reduce the effects of atopic dermatitis, Crohn's disease, constipation as well as candidiasis and certain genitourinary tract infections such as bacterial vaginosis, vaginitis, or urinary tract infections. The immunomodualting action of probiotics is helpful in reduction of allergic reactions, stimulation of phagocytosis by peripheral blood leukocytes and secretory IgA, modulation of cytokine gene expression, and many other immunological effects.
PCT patent No. WO 00/75284 by Olshenitsky et. al. describes a probiotic composition comprising a volatile fraction of a plant extract prepared by steam distillation and suspended microorganism such as E-coli. E-coli is not exactly non-pathogenic and may cause some harm to humans in certain conditions. No fermentation process of medium ingredients with bacteria is described in arriving at the end product. Rather, evaporation and condensation is used which limits the end properties of the product. For example, without E-coli the product looses its antagonistic activity. Even with E-coli present, the antagonistic activity is limited because some pathogens can still grow in the medium during incubation for 24 hours.
Probiotic preparations currently on the market appear in various forms: in dairy products, processed into a product such as chewing gum, pills, capsules, etc., suspended in milk, freeze-dried or air-dried. They are generally composed of large numbers of one or more bacterial species that are common constituents of normal intestinal flora. Fermented milk (yogurt) and cheese are the most common foods with probiotics. U.S. Pat. No. 6,228,358 by Toba describes an antioxidation product made from fermented milk. Zhang describes red rice fermentation products in the U.S. Pat. No. 6,046,022. Other forms of probiotic preparations are freeze-dried or air-dried and they are available in tablets and in capsules. U.S. Pat. No. 5,702,927 by Murofushi describes bacteria containing xanthan gum. In some cases, probiotics have been suspended in an appropriate milieu for better survival. For example, U.S. Pat. No. 5,908,622 by Barclay describes growing of microflora in fermentation medium containing certain sodium salts. U.S. Pat. No. 6,294,166 by Hsia describes a method of stabilization of specifically dried bacterial compositions mixed with specific nutrients, yeast and soy protein, for long periods of time. Some authors, for example, U.S. Pat. No. 6,203,797 by Perry; U.S. Pat. No. 6,080,401 by Reddy; and U.S. Pat. No. 5,171,575 by Shibata, used various food/herb compositions with probiotics, without fermentation, to enhance medicinal effects.
Fermented cultures containing microorganisms can be used in other industries such as in cosmetics and pharmaceutical industry. U.S. Pat. No. 6,270,811 by Fregonese describes a composition containing a microbial culture for skin regenerating and removing scars and wrinkles.
Despite their health promoting effects, probiotics have only demonstrated short-term effects. In the study of the health effects of probiotics, the incidence and/or duration of acute, short term diseases, such as diarrhea, are frequently measured. The effects of probiotic bacteria on the incidence of diseases with a protracted etiology, such as cancer or heart disease, have generally not been measured. Moreover, the effects of probiotics in life-threatening diseases, such as cancer for example, are doubtful. For probiotics to have their therapeutic effect they should be used in high doses daily and the duration of their use should be sufficiently long.
Importantly, attention has been focused on the microorganisms per se, not their products of metabolism. Lactic acid fermentation is mainly considered for dairy products. Only in some oriental foods such as cassava, mixtures of grains and legumes, have lactic acid fermentation been used for the preparation of a variety of foods made from raw materials of plant and animal origin. Processed food tends to loose a substantial part of its useful components, ferments for example, as compared to raw materials.
U.S. Pat. No. 5,292,511 by Kim describes the lactic acid fermentation process being used for aloe preservation, and the end product used as a health-food supplement. The product and process described in the patent is limited in time (up to 96 hours) and temperature of fermentation (20-35 degrees C.). At 40 degrees C. the product is reported to start to decompose. The inventors of the present invention believe that the fermentation process is not complete from the point of view of the instant invention.
U.S. Pat. No. 4,298,620 by Hagiwara proposes a fermentation process for obtaining a fermented tear grass product combining a water extract of tear grass with a Lactobacillus strain, and foods and feeds comprising that product. This patent is incorporated herein in its entirety. Importantly, one critical step in the process as described in this patent is heating of the tear grass before fermenting it. In our opinion, this step effectively damages all useful ferments contained in the grass and significantly reduces its effectiveness. Also, since the number of bacteria is not reduced at the end of cultivation, the acidity of the end product (as measured by concentration of lactate) is low at about 0.7 to 3%. Another limitation is the typical addition of sugar at the end of cultivation. Finally, a heat sterilization process at 80 degree C. for 40 minutes effectively destroys all live microorganisms and active ingredients, ferments for example.
Other fermentation patents of interest include U.S. Pat. No. 5,219,597 by Mok; U.S. Pat. No. 5,700,684 by Ehret; U.S. Pat. No. 6,156,320 by Izvekova; U.S. Pat. No. 5,556,785 by Kishida; U.S. Pat. No. 5,747,020 by Rutherford; U.S. Pat. No. 4,407,828 by Raccach; U.S. Pat. No. 3,963,835 by Gryczka; U.S. Pat. No. 4,018,650 by Busta; U.S. Pat. No. 4,528,199 by Moon; U.S. Pat. No. 4,579,740 by Matrozza; U.S. Pat. No. 4,897,350 by El-Megeed; U.S. Pat. No. 4,749,652 by Robinson; U.S. Pat. No. 4,816,267 by Oka; U.S. Pat. No. 4,855,147 by Yokota; U.S. Pat. No. 4,579,739 by Darbyshire; U.S. Pat. No. 4,664,919 by Yan; U.S. Pat. No. 4,770,882 by Ingouf; and U.S. Pat. No. 3,944,676 by Fridman. They depict mostly various fermentation processes that are somewhat similar to the subject of the invention but in most cases these processes are short-term or carried out at low temperatures in solid phase and therefore incomplete from the point of view of the present invention.
One probable reason for limited effectiveness of probiotics in general is because of poor binding of the active microorganisms to the internal linings or external surface of the human body. Bacteria, especially in the state of freeze-dried suspension, have only limited time to develop a bond with the host. It takes several hours for the bacteria to become active after being consumed. Therefore, the bacteria are frequently expelled by natural processes such as digestion without allowing it to bond to the intestines and to produce enzymes, vitamins, amino acids, organic acids and other products of their metabolism. It is these metabolic products that represent the ultimate goal of the application of microorganisms. Live microorganisms might have a better chance to remain on the surface and tissue lining and attach thereto.
The need therefore exists for a medium containing live microorganisms as well as their metabolic products in high concentrations. Its application for humans is believed to be more effective and provide long-term benefits than the presently known suspensions of such microorganisms mostly in inactive state, even consumed in a high concentration.